RU2592282C1 - Method of producing nonapeptides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a solid-phase method of producing nonapeptides of the following formula I-III: $$R-Ar{g}^1-Ly{s}^2-Ly{s}^3-Ty{r}^4-Ly{s}^5-Ty{r}^6-Ar{g}^7-Xa{a}^8-Ly{s}^9-N{H}_2,$$

where R=H, Xaa=L-Arg (I); R=Me, Xaa=L-Arg (II); R=H, Xaa=D-Axg (III). Nonapeptides synthesis as per formula I-III is carried out by serial growth of the peptide chain, starting with the C-terminal amino acid covalently bound with the polymer matrix using N^α-protected derivatives of arginine, for blocking the guanidine function of which protonating is used. For the guanidine function protonation 1-hydroxybenzotriazole, hexafluorophosphate or tetrafluoroborate is used. Obtained nonapeptidilpolymer is processed with an unblocking agent for removing the protective groups and the polymeric matrix and in 1 stage the end product is given out by means of HELC.

EFFECT: method increases the output of end products, simplifies and lowers the cost of their production process.

4 cl, 5 dwg, 5 tbl, 3 ex

Description

The invention relates to the field of physiologically active peptides, and in particular to a method for producing nonapeptides containing residues of L-, D- or L- N ^α- methyl-arginine of the formula I-III in the molecule:

R-Arg ¹ -Lys ² -Lys ³ -Tyr ⁴ -Lys ⁵ -Tyr ⁶ -Arg ⁷ -Xaa ⁸ -Lys ⁹ -NH ₂ ,

Where

R = H, Xaa = L-Arg (I)

R = Me, Xaa = L-Arg (II)

R = H, Xaa = D-Arg (III)

Compounds (I-III) are peptide inhibitors of myosin light chain kinase (MLCK) and have the ability to regulate changes in the permeability of epithelium and vascular endothelium [Sekridova A.V., Sidorova M.V., Azmuko A.A., Molokoedov A.S. , Bushuev V.N., Marchenko A.V., Shcherbakova O.V., Shirinsky V.P., Bespalova Zh.D. Proteinase resistant peptide inhibitors of myosin light chain kinase. Bioorganic chemistry. - 2010, 36 (4), p. 498-504]. Peptide (I) in vitro is capable of influencing intestinal epithelial permeability [Clayburgh D.R., Barrett T.Α., Tang Y., Meddings J.B., Van Eldik L.J., Watterson D.M., Clarke L.L., Mrsny R.J., Turner J.R. Epithelial myosin light chain kinase-dependent barrier dysfunction mediates Τ cell activation-induced diarrhea in vivo. J. Clin. Invest. 115 (10): 2702-2715. 2005], peptide (II) [RF Patent No. 2402565, IPC C07K 7/00, publ. October 27, 2010] and peptide (III) [RF Patent No. 2493164, IPC C07K, publ. September 20, 2013] have the ability to prevent an increase in the permeability of the vascular endothelium and can be used as a means of reducing the pathological hyperpermeability of the vascular endothelium in various fields of medicine (in cardiology, toxicology, neurosurgery, oncology, etc.).

A known method of producing peptides of the formula (I-III) by solid-phase method. In the process of solid-phase synthesis of peptides (TFS), the synthesized polypeptide chain is covalently attached to an insoluble inert polymer matrix. The selection of the target product at each stage of TFS is carried out by appropriate washing and filtration of the peptidyl polymer. The synthetic protocol includes several successive chemical transformations - stages that are repeated in each synthesis cycle (synthesis cycle - addition of one amino acid). The standard synthesis cycle includes the following main stages: 1) release — removal of the N ^α -protective guppa (Fmoc) by treatment of the peptidylpolymer with a solution of a secondary amine (piperidine, 4-methylpiperidine, 1,8-diazabicyclo [5.4.0] undec-7-ene and t .p.) in a suitable solvent; 2) obtaining an activated derivative of an attached amino acid; 3) condensation - addition of the remainder of the Fmoc-protected amino acid to the peptidylpolymer due to the formation of an amide bond. Between the main steps, the peptidyl polymer is washed with an organic solvent to remove excess of the corresponding reagents. The synthesis of peptides (I-III) was carried out by stepwise extension of the peptide chain, starting from the C-terminal amino acid on the polymer matrix, using 4-fold excesses of the corresponding protected Fmoc amino acids in the presence of a condensing agent, followed by the release of the final peptidyl polymer in 2 stages: treatment with a solution secondary base (piperidine or others) to remove the Fmoc-protection and then trifluoroacetic acid with special additives for 16 hours, followed by purification of the obtained product using high hydroelectric liquid chromatography (HPLC). In FIG. 1 shows a synthesis scheme for nonapeptides corresponding to known methods. When TFS peptides (I-III), the tactics of maximum protection of the side functions of amino acids were used. With TPS of the peptide (I), the guanidine groups of arginine residues were protected with the 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) group. The total yield of the peptide (based on the starting amino acid) was 54.8%. In the synthesis of peptides II and III, the guanidine groups of the N-terminal N ^α -methyl-substituted Arg ¹ residue were protected with 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) protection, the side groups of Arg ^7.8 residues were blocked with Pmc protection. In this case, peptides of formula II and III were obtained with yields of 38.8 and 47.1%, respectively, based on the starting amino acid attached to the polymer. Table 1 shows a comparative assessment of the known and claimed methods of solid-phase synthesis of nonapeptides (I-III).

The disadvantages of the known methods are the use of expensive and inaccessible derivatives of arginine, the difficulty of cleaving arginine protections after synthesis (a long time of 16 hours and adverse reactions associated with the cleavage of the arylsulfonyl protections of the guanidine group of Arg residues), insufficiently high yield of the target product and multi-stage process.

The above disadvantages make the known method unsuitable for large-scale synthesis of nonapeptides of the formula (I-III).

The objective of the invention is to provide a method for producing nonapeptides, which simplifies the procedure for the synthesis of gram quantities of nonapeptides of the formula I-III, leading to: 1) the absence of adverse reactions when removing the arylsulfonyl protections of the guanidine function of arginine residues, 2) reducing the time of the final release of peptides, 3) reducing the cost of the derivatives used amino acids, 4) simplifying the purification of the crude product.

The technical result consists in simplifying the method.

The technical result is achieved by the fact that the solid-phase synthesis of nonapeptides of the formula I-III:

R-Arg ¹ -Lys ² -Lys ³ -Tyr ⁴ -Lys ⁵ -Tyr ⁶ -Arg ⁷ -Xaa ⁸ -Lys ⁹ -NH ₂ ,

Where

R = H, Xaa = L-Arg (I)

R = Me Xaa = L-Arg (II)

R = H, Xaa = D-Arg (III)

carried out by sequentially increasing the peptide chain on a polymer matrix to obtain the corresponding nonapeptidylpolymer using arginine derivatives with unprotected guanidine function.

The implementation of the method

Upon receipt of nonapeptides by the claimed method, the iV-terminal arginine residue is added as N ^α- Boc (tert-butyloxycarbonyl) -X-Arg-OH, where X = H (I), X = CH ₃ (II), (III). Residues Z-Arg ⁷ and L-Arg ⁸ (I) and (II) or D-Arg ⁸ (III) are introduced into the peptide chain in the form of the corresponding Fmoc derivatives. In FIG. 2 shows a scheme for the synthesis of nonapeptides (II) - (III) in accordance with the claimed method. Used arginine derivatives are inexpensive commercially available products that can be obtained in one step by conventional methods of organic chemistry in solution. During TFS, at the stage of addition of the corresponding residues of L-, D- and N ^α- Me-L-arginine, temporary blocking of its lateral guanidine function by protonation (salt formation) is carried out.

Chlorine and bromohydrates are used to protest the guanidine group in the synthesis of peptides in solution.

1-hydroxybenzotriazole effectively protonates the guanidine group to form salts that are readily soluble in the solvents used for TPS. In the inventive method, the addition of N ^α -Fmoc-L-Arg-OH or N ^α -Fmoc-D-Arg-OH is carried out in the presence of 2-3 equivalents of HOBt with respect to the Fmoc amino acid and an equivalent amount of Ν, Ν′-diisopropylcarbodiimide. The use of 1-hydroxybenzotriazole (HOBt) in solid-phase synthesis (TFS) of peptides is widely known. HOBt significantly increases the rate of acylation reaction with carbodiimides, effectively inhibits racemization and the formation of N-acylurea.

Another important advantage of the proposed method is the possibility of using condensing agents based on phosphonium / uronium salts, such as benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), 2- (1H-benzotriazol-1-yl) -1,1 , 3,3-tetramethyluronium hexafluorophosphate (HBTU) or tetrafluoroborate (TBTU) without the use of an organic base. Typically, when using these reagents in TPS, 2-3 equivalents of an organic base (diisopropylethylamine or N-methylmorpholine) necessary for binding acidic counterions - hexafluorophosphate or tetrafluoroborate - are used to activate the carboxyl group. Excess bases are known to lead to racemization of the attached amino acid. It turned out that the free guanidine group effectively plays the role of an acceptor of acidic counterions and at the same time it blocks. Thus, in the case of activation of the carboxyl group of Fmoc-Arg-OH using phosphonium / uronium salts, its guanidine group (pK _a 12.5) plays the role of an organic base. An additional advantage of the claimed method is the fact that the activated derivative of arginine is obtained in situ under a continuous process that does not require the introduction of an additional step in the preparation of activated derivatives of arginine.

Since nonapeptides I-III contain three arginine residues per molecule, during the release of the α-amino group in arginine-containing peptidyl polymers, starting with the dipeptidyl polymer, not only the α-amino group is released, but the guanidine function of the arginine residues already existing in the polymer chain is also deprotonated . Subsequently, upon addition of the following amino acid, regardless of the chosen condensation method, not only the α-amino group, but also the guanidine group of arginine enters the acylation reaction. As a result, by-products are formed that not only lower the yield of the target substance, but also significantly complicate its purification.

To prevent the side reaction of acylation of the guanidine function of arginine residues, an additional protonation of arginine is necessary after each stage of the release of arginine-containing peptidyl polymers. Additional treatment of the peptidylpolymer with a 5% solution of HOBt in Ν, Ν-dimethylformamide, Ν, Ν-dimethylacetamide or N-methypyrrolidone for 5 minutes before condensation fully protects the guanidine group of arginine residues by protonation and does not interfere with the acylation of the α-amino group of the peptide . HOBt is a neutral molecule, highly soluble in organic solvents, and these solutions are storage stable.

From a practical point of view, this additional washing organically fits into the technological process of TPS, which is important when scaling synthesis to obtain peptides in large quantities.

At the end of the synthesis, the obtained nonapeptidyl polymer is treated with a deblocking mixture, all protecting groups and the polymer matrix are cleaved in one step, and the desired product is isolated by high performance liquid chromatography. The purity of the obtained peptides is determined by reverse phase HPLC, the structure of the peptides is confirmed by ¹ H-NMR spectroscopy and mass spectrometry. A comparative assessment of the yields and purity of the peptides obtained by the described and claimed methods is represented by graphic materials. Table 1 presents a comparative assessment of the known and claimed methods of solid-phase synthesis of nonapeptides (I-III). In FIG. 3 shows the analytical HPLC profile of the crude product of solid-phase synthesis of nonapeptide (I) H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH ₂ (a) in a known manner, (b) the claimed method. The content of the target peptide (I) is 55% (a) and 70% (b), respectively. Kromasil C18 column (4.6 × 250 mm), particle size 5 μm, mobile phase: buffer A 0.05 M KH ₂ PO ₄ , pH 3, buffer B 70% acetonitrile + 30% buffer A, gradient B from 0 to 60% for 30 min, the elution rate of 1 ml / min. In FIG. 4 shows the analytical HPLC profile of the crude product of solid-phase synthesis of nonapeptide (III) H-MeArg-Lys-Lys-Tyr-Lys-Tyr-Arg-DArg-Lys-NH ₂ (a) by a known method, (b) by the claimed method. The content of peptide (III) is 76% (a) and 83% (b), respectively. Kromasil C18 column (4.6 × 250 mm), particle size 5 μm, mobile phase: buffer A 01% TFA, buffer B - 80% acetonitrile + 20 buffer A, gradient B from 0 to 60% in 30 min, elution rate 1 ml / min

The method is illustrated by the following examples.

Example 1. The synthesis of the peptide H-Arg ¹ -Lys ² -Lys ³ -Tyr ⁴ -Lys ⁵ -Tyr ⁶ -Arg ⁷ -Arg ⁸ -Lys ⁹ -NH ₂ (I)

Derivatives of amino acids (AA) and hexafluorophosphate (benzotriazol-1-yl) hydroxy tris (dimethylamino) phosphonium (BOP) NovaBiochem, Bachem, Switzerland), Ν, Ν′-diisopropylcarbodiimide (DIC), Ν, Ν-diisopropyl were used DIPEA), hydroxybenzotriazole (HOBt), triisobutylsilane (TIBS) company Fluka, Switzerland, 4-methylpiperidine (4-MePip, Aldrich, USA). For the synthesis, N-methylpyrrolidone, dichloromethane (DCM), trifluoroacetic acid (TFA) from Fluka, Switzerland were used; for chromatography, acetonitrile (Panreac, Spain). Analytical high performance liquid chromatography (HPLC) was carried out on a chromatograph (Gilson, France), a Kromasil C18 column, 5 μm, (4.6 × 250 mm) (Akzo Nobel, USA) was used, buffer A - 0.05 M KH ₂ HO _{4 was} used as eluents , pH 3, buffer B — 70% acetonitrile in buffer A (condition 1) or buffer A — 0.1% TFA, buffer B — 80% acetonitrile in buffer A (condition 2), elution was carried out with a concentration gradient of buffer B in buffer A from 0 % to 60% in 30 minutes A flow rate of 1 ml / min, detection at 220 nm. The structure of the obtained peptides was proved by ¹ H-NMR spectra and mass spectrometry data. ¹ H-NMR spectra were recorded on a WM-500 spectrometer (Bruker) 500 MHz (Germany) in deuterated dimethyl sulfoxide (DMSO-d ₆ ) at 300 K, the concentration of peptides was 2-3 mg / ml. Chemical (δ, ppm) shifts were measured relative to tetramethylsilane. Mass spectra were recorded on an UltraflexTOF / TOF instrument (Bruker Daltonics, Germany) with a time-of-flight base using the MALDI method. For solid-phase synthesis, a copolymer of styrene with 1% divinylbenzene with 4- (2,4-dimethoxyphenyl) -9-fluorenylmethoxycarbonyl-aminomethyl-phenoxy-anchor group (Rink-amide-polymer) from Nova BioChem, Switzerland, designed to produce peptide amides containing 0.64 was used mmol / g of amino groups. The nonapeptide amide was synthesized from the C-terminus, stepwise (adding one amino acid each), starting from 1.0 g (0.64 mmol) of the Rink-amide polymer in accordance with the protocol. For addition of all amino acids, with the exception of arginine, 4-fold excess of acylating agents was used once. The guanidine groups of residues Arg ^{1,7,8 were} blocked by protonation. Table 2 presents the protocol of solid-phase synthesis of the peptide (I). Upon addition of arginine residues, condensation was performed twice using 2-fold excess of acylating agents. The completeness of the reaction was monitored using a test for free amino groups in peptidyl polymers with ninhydrin.

The final unblocking and cleavage of the nonapeptide from the polymer was carried out in one step by treating the corresponding nonapeptidyl polymer with a mixture of 20 ml of TFA, 0.5 ml of deionized water and 0.5 ml of TIBS for 2 hours, then the polymer was filtered off, washed with 2 × 5 ml of the deblocking mixture, the filtrate was evaporated and the residue dry ether was added. The precipitate was filtered off, washed with DCM (3 × 10 ml), ether (3 × 10 ml), dried in a vacuum desiccator. Obtained 0.82 g of a crude solid-phase synthesis product with a basic substance content of 72%. The substance was purified in portions using preparative HPLC on a Beckman instrument (USA) using a 130T Diasorb C16 column (25 × 250 mm), sorbent particle size 10 μm. As eluents used buffer A - 0.01 M solution of ammonium acetate and buffer B - 80% acetonitrile in water. Elution was performed with a gradient of 0.5% per minute of buffer B in buffer A from 100% of buffer A at a rate of 10 ml / min. Peptides were detected at a wavelength of 220 nm. Fractions containing the desired product were combined, acetonitrile was evaporated and lyophilized. As a result, 0.51 g (61.2% based on the starting amino acid) of the nonapeptide amide (I) was obtained. Product homogeneity determined by analytical HPLC was 97.4%. Mass spectrum: 1325.1, calculated 1324.6.

Example 2. H- (N-Me) -Arg ¹ -Lys-Lys-Tyr-Lys-Tyr-Arg ⁷ -Arg ⁸ -Lys-NH ₂ (II)

The synthesis of the nonapeptide (II) amide was carried out similarly to the synthesis of the peptide (I) according to Scheme 2, stepwise (adding one amino acid each), starting from 1.56 g (1.0 mmol) of the Rink-amide polymer. Table 3 presents the protocol of solid-phase synthesis of the peptide (II). For addition of all amino acids, with the exception of arginine, 3-fold excess of acylating agents was used once. The guanidine groups of Arg residues were blocked by protonation (see protocol). Upon addition of arginine residues, condensation was performed twice using 1.5-fold excess of acylating agents. The completeness of the reaction was monitored using a test for free amino groups in peptidyl polymers with ninhydrin.

The final release of nonapeptide (II) was carried out in one step by treating the corresponding nonapeptidyl polymer with a mixture of 30 ml of TFA, 0.75 ml of deionized water and 0.75 ml of TIBS for 2 hours. Then the polymer was filtered off, washed with 2 × 7 ml of the release mixture, the filtrate was evaporated and the residue was added dry ether. The precipitate was filtered off, washed with DCM (3 × 10 ml), ether (3 × 15 ml), dried in a vacuum desiccator. 1.24 g of a solid solid synthesis crude product with a basic substance content of 70% was purified in portions using preparative HPLC as described in Example 1. As a result, 0.715 g (53.4% based on the starting amino acid) of nonapeptide (II) amide was obtained. The homogeneity of the product, determined by analytical HPLC under conditions 1 and 2, is 97.5%. Mass spectrum: 1338.9, calculated 1338.7.

Example 3. H- (N-Me) -Arg ¹ -Lys-Lys-Tyr-Lys-Tyr-Arg ⁷ -D-Arg ⁸ -Lys ⁹ -NH ₂ (III)

The synthesis of the nonapeptide (III) amide was carried out similarly to the synthesis of the peptide (I) according to Scheme 2, stepwise (adding one amino acid each), starting from 3.12 g (2.0 mmol) of the Rink-amide polymer. Table 4 presents the protocol of solid-phase synthesis of the peptide (III). For addition of all amino acids, with the exception of arginine, 3-fold excess of acylating agents was used once. The guanidine groups of Arg residues were blocked by protonation (see protocol). Upon addition of arginine residues, condensation was performed twice using 1.5-fold excess of acylating agents. The completeness of the reaction was monitored using a test for free amino groups in peptidyl polymers with ninhydrin.

The final release and cleavage of the nonapeptide from the polymer was carried out in one step by treating the corresponding nonapeptidyl polymer with a mixture of 60 ml of TFA, 1.5 ml of deionized water and 1.5 ml of TIBS for 2 hours. Then the polymer was filtered off, washed with 2 × 15 ml of the deblocking mixture, the filtrate was evaporated and the residue dry ether was added. The precipitate was filtered off, washed with DCM (3 × 20 ml), ether (3 × 30 ml), dried in a vacuum desiccator. The crude solid-phase synthesis product (2.48 g) with a basic substance content of 83% was purified in portions. As a result, 1.62 g (60.5% based on the starting amino acid) of the nonapeptide (III) amide were obtained. The homogeneity of the product, determined by analytical HPLC under conditions 1 and 2, is 97.9%. Mass spectrum: 1339.0, calculated 1338.7. In FIG. 5 shows the data of ¹ H-NMR spectroscopy. Table 5 shows the chemical shifts (δ, ppm) of the proton signals of the nonapeptide (III) amide.

Claims (4)

1. The method of producing nonapeptides of the formula I-III:
R-Arg ¹ -Lys ² -Lys ³ -Tyr ⁴ -Lys ⁵ -Tyr ⁶ -Arg ⁷ -Xaa ⁸ -Lys ⁹ -NH ₂ ,
Where
R = H, Xaa = L-Arg (I)
R = Me, Xaa = L-Arg (II)
R = H, Xaa = D-Arg (III)
solid-phase method by sequentially increasing the peptide chain, starting with the C-terminal amino acid covalently linked to the polymer matrix, subsequent processing of the obtained nonapeptidyl polymers with a deblocking agent to remove the protective groups and the polymer matrix and isolate the final product using high-performance liquid chromatography (HPLC), characterized in that protonation is used to block the guanidine function of L-, D-, and Me- L-arginine residues. 2. The method according to p. 1, characterized in that for the protonation of the guanidine group in peptidyl polymers, an additional washing with a 5% solution of 1-hydroxybenzotriazole is used. 3. The method according to p. 1, characterized in that the condensation of arginine residues is repeated twice using half the amount of acylating agents, and BOP, HBTU or TBTU are used as the condensing agent without the use of an organic base. 4. The method according to p. 1, characterized in that the condensation of arginine residues is repeated twice using 2-fold excess of amino acid derivatives, 2-fold excess of Ν, Ν′-diisopropylcarbodiimide and 4-6-fold excess of HOBt with respect to the content of amino groups in peptidyl polymers.

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